Development of a male contraceptive could in principle be achieved through perturbation of the reproductive endocrine axis, disruption of spermatogenesis in the testis, inhibition of sperm maturation in the epididymis, or alteration of sperm structure or function during ejaculation. Within the testis one could imagine blocking the initiation of spermatogenesis, the progression through meiosis, or the terminal differentiation of spermatids. Genetic models for the feasibility of some of these scenarios currently exist. For this proposal we have chosen to expand the basic knowledge of spermatogonial stem cells under the belief that in the future it might be feasible to develop contraceptive agents that reversibly block the initiation of spermatogenesis. Continued spermatogenesis in mammals requires maintaining an exquisite balance between the self-renewal and differentiation of a small population of spermatogonial stem cells (SSCs). Until recently almost nothing was known about the maintenance of SSC self-renewal. Rapid advances in this exciting area have revealed that SSCs respond to at least two independent extrinsic signaling pathways emanating from Sertoli cells. Several intrinsic signals help maintain SSC homeostasis. An unexpected discovery has been the realization that adult SSCs can readily convert to a pluripotent state that appears very much like that of an embryonic stem cell, offering the promise that SSCs may be useful in regenerative medicine. During the previous funding period we showed that the promyelocytic leukemia zinc finger protein, Plzf, is expressed in SSCs and required for their self-renewal. Loss of Plzf function shifts the balance between stem cell self-renewal and differentiation, toward differentiation at the cost of self-renewal. Plzf is a transcriptional represser that has been shown to associate with members of the N-Cor2/SMRT co-repressor complex, and with Bmi1, a Polycomb group protein and component of the Polycomb repressive complex 1. Both of these complexes confer epigenetic modifications to chromatin. Our hypothesis is that Plzf recruits members of the Ncor-2/SMRT co-repressor complex, and perhaps components of the Polycomb group repressive complex 1, to pluripotency and/or differentiation genes in SSCs. Loss of Plzf creates an unstable epigenetic state that compromises self-renewal. A full understanding of how Plzf functions to maintain SSC self-renewal will require identification of the transcriptional targets of Plzf, investigation of the molecular mechanism by which Plzf represses transcription in SSCs, and characterization of the various signaling pathways in the SSC niche that converge to regulate Plzf and the other intrinsic factors required for SSC self-renewal. The three Specific Aims in this proposal attempt to address each of these issues.